PROJECT SUMMARY: Despite a decade of engineering advancements and clinical process improvements, 1 million healthcare- associated infections in the U.S. can be attributed to indwelling medical devices annually. Zinc oxide nanoparticles (ZnO-NPs) are one of the most promising emerging antimicrobials with potential to combat device related infection. ZnO-NPs are inexpensive, stable, and easy to prepare with broad antimicrobial spectrum and wide therapeutic window. However, the antimicrobial mechanism of action of ZnO-NPs remains elusive. This proposal is specifically motivated to better understand the mechanism of action of ZnO-NPs. Such understanding is necessary to guide the design of device coatings that preserve antibacterial function in vivo. Reactive oxygen species (ROS) generation or membrane disruption are hypothesized mechanisms of action. However the literature is inconsistent and our preliminary data suggests that these NP effects are not sufficient. We recently demonstrated that ZnO-NPs have shape-dependent, biomimetic, reversible, enzyme inhibition properties. The central research question for this career development grant is: To what extent does ZnO-NP behavior as an enzyme inhibitor contribute to antimicrobial activity? I have multidisciplinary training in medicine, engineering, and molecular biology that is well-suited to address this question. My ultimate career goal is to become a clinician-scientist. I plan to have a clinical interest in sepsis as it relates to indwelling medical devices and an independently funded research program focused on the development of novel biomaterials to resist microbial contamination and infection. This proposal was developed to solidify my expertise, formalize my research niche, and garner the resources for the next phase of career development. My specific career development objectives for the next four years are to: 1. Solidify my expertise in microbiology (including biofilm microbiology), microbial-surface interaction, nanoparticle technology, and translational research. 2. Master techniques in evaluating mechanisms of action of antimicrobial and anti-biofilm materials. 3. Generate sufficient preliminary data and publication record to obtain independent research funding. 4. Secure my niche as an expert in bacterial-nanomaterial interactions. 5. Obtain secondary appointment in the College of Engineering so that I can work with and mentor graduate students in their research and career development. I have assembled a mentorship team of experts co-localized at the University of Michigan North Campus Research Complex with experience in clinical medicine, microbiology, material science and engineering, and product development/commercialization. Together we have devised a highly-individualized, project-oriented training plan that includes regular mentorship meetings, formal didactic education, career development workshops, and presentation at local and national conferences. Partnered with this career development plan is an innovative research plan. By synthesizing ZnO-NPs that are identical in surface chemistry but differ only in shape we can control the potential for enzyme inhibition and address the central research question above. Using these novel preparations, we will test the hypothesis: Pyramidal ZnO-NPs inhibit a cohort of bacterial enzymes which are critical to survival. Our research specific aims are to: 1. Quantify aerobic metabolism, membrane integrity, and microbial death in a commonly isolated medical device pathogen (i.e., Staphylococcus aureus) as a function of exposure time to spherical vs pyramidal ZnO-NPs. 2. Identify genes involved in enzyme inhibition by ZnO-NPs using a mariner transposon mutant library of S. aureus. 3. Determine the subset of S. aureus proteins that specifically complex with ZnO-NPs in a shape- dependent manner by 2D-gel electrophoresis followed by liquid chromatography paired with tandem mass spectroscopy (LC-MS/MS).